ANA Profile 8 ELISA. 12x8

Transcription

1 Instructions for use ANA Profile 8 ELISA Enzyme immunoassay for the qualitative determination of individual IgG antibodies against dsdna, RNP, Sm, SS-A/Ro, SS-B/La, Scl-70, CENP-B and Jo-1 in human serum. RE x8 2-8 C I B L I N T E R N A T I O N A L G M B H Flughafenstrasse 52a Phone: +49 (0) IBL@IBL-International.com D Hamburg, Germany Fax: +49 (0)

2 1. INTENDED USE Enzyme immunoassay for the qualitative determination of individual IgG antibodies against dsdna, RNP, Sm, SS-A/Ro, SS-B/La, Scl-70, CENP-B and Jo-1 in human serum. 2. SUMMARY AND EXPLANATION Circulating autoantibodies against various intracellular antigens (antinuclear antibodies, ANA) are characteristic for systemic, autoimmune-mediated rheumatic diseases. These comprise systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), Sjögren's syndrome (SS), progressive systemic sclerosis (PSS, scleroderma)/crest syndrome, rheumatoid arthritis (RA) and polymyositis (PM). Often, these disorders can be diagnosed by measuring their associated antibodies. Eight antigens specifically recognised by these antibodies are immobilised separately on the solid phase of the present enzyme-linked immunosorbent assay (ELISA): Antigen Source Disease dsdna plasmid SLE RNP (proteins A, C, 68kDa) human, recombinant MCTD, SLE, RA, PSS Sm (proteins B, B', D) bovine thymus SLE SS-A/Ro (60kDa-protein) bovine thymus SS, SLE SS-B/La human, recombinant SS, SLE Scl-70 (DNA-topoisomerase I) human, recombinant PSS CENP-B human, recombinant PSS (CREST) Jo-1 (Histidyl-tRNA-synthetase) human, recombinant PM 3. TEST PRINCIPLE Solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle. The wells are coated each with one of the antigens mentioned above. Specific antibodies of the sample binding to the antigen coated wells are detected by a secondary enzyme conjugated antibody (E-Ab) specific for human IgG. After the substrate reaction the intensity of the color developed is proportional to the amount of IgGspecific antibodies detected. Results of samples are estimated qualitatively using a cut-off. 4. WARNINGS AND PRECAUTIONS 1. For in-vitro diagnostic use only. For professional use only. 2. Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood. 3. In case of severe damage of the kit package please contact IBL or your supplier in written form, latest one week after receiving the kit. Do not use damaged components in test runs, but keep safe for complaint related issues. 4. Observe lot number and expiry date. Do not mix reagents of different lots. Do not use expired reagents. 5. Follow good laboratory practice and safety guidelines. Wear lab coats, disposable latex gloves and protective glasses where necessary. 6. Reagents of this kit containing hazardous material may cause eye and skin irritations. See MATERIALS SUPPLIED and labels for details. Material Safety Data Sheets for this product are available on the IBL- Homepage or upon request directly from IBL. 7. Chemicals and prepared or used reagents have to be treated as hazardous waste according to national biohazard and safety guidelines or regulations. 8. Avoid contact with Stop Solution. It may cause skin irritations and burns. 9. Some reagents contain sodium azide (NaN 3 ) as preservatives. In case of contact with eyes or skin, flush immediately with water. NaN 3 may react with lead and copper plumbing to form explosive metal azides. When disposing reagents, flush with a large volume of water to avoid azide build-up. 10. All reagents of this kit containing human serum or plasma have been tested and were found negative for anti-hiv I/II, HBsAg and anti-hcv. However, a presence of these or other infectious agents cannot be excluded absolutely and therefore reagents should be treated as potential biohazards in use and for disposal. V / 7

3 5. STORAGE AND STABILITY The kit is shipped at ambient temperature and should be stored at 2-8 C. Keep away from heat or direct sun light. The storage and stability of specimen and prepared reagents is stated in the corresponding chapters. The microtiter strips are stable up to the expiry date of the kit in the broken, but tightly closed bag when stored at 2-8 C, together with the desiccant. 6. SPECIMEN COLLECTION AND STORAGE Serum The usual precautions for venipuncture should be observed. It is important to preserve the chemical integrity of a blood specimen from the moment it is collected until it is assayed. Do not use grossly hemolytic, icteric or grossly lipemic specimens. Samples appearing turbid should be centrifuged before testing to remove any particulate material. Storage: 2-8 C -20 C Keep away from heat or direct sun light. Stability: 3 d > 3 d Avoid repeated freeze-thaw cycles. 7. MATERIALS SUPPLIED Quantity Symbol Component 1 x 12 x 8 MTP Microtiter Plate Each strip coated well by well with one of the eight individual antigens mentioned above. Enzyme Conjugate IgG 1 x 14 ml ENZCONJ IgG Ready to use, red colored. Contains: anti-human IgG, conjugated to peroxidase, protein-containing buffer and preservatives. 1 x 3 ml CONTROL + Positive Control (= Calibrator) Ready to use, red colored. Contains: Specific IgG antibodies, TBS and preservatives. 1 x 3 ml CONTROL - Negative Control Ready to use, green colored. Contains: TBS and preservatives. 1 x 100 ml SAMPLEDIL Sample Diluent Ready to use, orange colored. Contains: TBS and preservatives. 1 x 14 ml TMB SUBS TMB Substrate Solution Ready to use, colorless. Contains: TMB, hydrogen peroxide. 1 x 100 ml WASHBUF CONC Wash Buffer, Concentrate (10x) Blue colored. Contains: TBS and preservatives. 1 x 14 ml STOP TMB Stop Solution Ready to use, colorless. 0.2 M H 2SO MATERIALS REQUIRED BUT NOT SUPPLIED 1. Micropipettes (Multipette Eppendorf or similar devices, < 3% CV). Volumes: 5; 100; 500 µl 2. Calibrated measures 3. Tubes (1 ml) for sample dilution 4. 8-Channel Micropipettor with reagent reservoirs 5. Wash bottle, automated or semi-automated microtiter plate washing system 6. Microtiter plate reader capable of reading absorbance at 450 nm (reference wavelength nm) 7. Bidistilled or deionised water 8. Paper towels, pipette tips and timer V / 7

4 9. PROCEDURE NOTES 1. Any improper handling of samples or modification of the test procedure may influence the results. The indicated pipetting volumes, incubation times, temperatures and pretreatment steps have to be performed strictly according to the instructions. Use calibrated pipettes and devices only. 2. Once the test has been started, all steps should be completed without interruption. Make sure that required reagents, materials and devices are prepared ready at the appropriate time. Allow all reagents and specimens to reach room temperature (18-25 C) and gently swirl each vial of liquid reagent and sample before use. Mix reagents without foaming. 3. Avoid contamination of reagents, pipettes and wells/tubes. Use new disposable plastic pipette tips for each component and specimen. Do not interchange caps. Always cap not used vials. Do not reuse wells/tubes or reagents. 4. Use a pipetting scheme to verify an appropriate plate layout. 5. Incubation time affects results. All wells should be handled in the same order and time sequences. It is recommended to use an 8-channel Micropipettor for pipetting of solutions in all wells. 6. Microplate washing is important. Improperly washed wells will give erroneous results. It is recommended to use a multichannel pipette or an automatic microplate washing system. Do not allow the wells to dry between incubations. Do not scratch coated wells during rinsing and aspiration. Rinse and fill all reagents with care. While rinsing, check that all wells are filled precisely with Wash Buffer, and that there are no residues in the wells. 7. Humidity affects the coated wells/tubes. Do not open the pouch until it reaches room temperature. Unused wells/tubes should be returned immediately to the resealed pouch including the desiccant. 10. PRE-TEST SETUP INSTRUCTIONS Preparation of Components Dilute / dissolve Component Diluent Relation Storage Stability 100 ml WASHBUF CONC ad 1000 ml bidist. water 1: C 4 weeks Dilution of Samples Sample to be diluted With Relation Remarks Serum generally SAMPLEDIL 1:101 e.g. 5 µl µl V / 7

5 11. TEST PROCEDURE 1. Immediately prior to use, wash the solid phase once: fill wells with 300 µl Wash Buffer each, soak for about 10 seconds in the wells and remove. 2. Pipette 100 µl of each Control and diluted sample (1-10) into the respective wells of the Microtiter Plate as depicted: 3. Incubate 30 min at room temperature (18-25 C). 4. Discard incubation solution. Wash plate 3 x with 300 µl of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel. 5. Pipette 100 µl of Enzyme Conjugate into each well. 6. Incubate 30 min at room temperature (18-25 C). 7. Discard incubation solution. Wash plate 3 x with 300 µl of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel. 8. For adding of Substrate and Stop Solution use, if available, an 8-channel Micropipettor. Pipetting should be carried out in the same time intervals for Substrate and Stop Solution. Use positive displacement and avoid formation of air bubbles. 9. Pipette 100 µl of TMB Substrate Solution into each well. 10. Incubate 30 min at room temperature (18-25 C) (protect from direct sunlight). 11. Stop the substrate reaction by adding 100 µl of TMB Stop Solution into each well. Briefly mix contents by gently shaking the plate. Color changes from blue to yellow. 12. Measure optical density with a photometer at 450 nm (Reference-wavelength: nm) within 30 min after pipetting of the Stop Solution. 12. QUALITY CONTROL The test results are only valid if the test has been performed following the instructions. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable standards/laws. The Positive and Negative Control must be found within the acceptable ranges as stated in the QC Certificate. If the criteria are not met, the run is not valid and should be repeated. Each laboratory should use known samples as further controls. In case of any deviation the following technical issues should be proven: Expiration dates of (prepared) reagents, storage conditions, pipettes, devices, incubation conditions and washing methods. It is recommended to participate at appropriate quality assessment trials. V / 7

6 13. CALCULATION OF RESULTS The test is evaluated in a qualitative manner. The absorbance (OD) of the samples is compared with the borderline OD (= cut-off), separately for each of the 8 parameters. The borderline OD is determined by means of the Positive Control which at the same time functions as Calibrator, according to the formula: OD borderline = OD positive control x factor The factor depends on the kit lot and is individually quoted for each parameter in the lot-specific QC Certificate which is included with each test kit. Example: OD positive control = factor = 0.35 OD borderline = x 0.35 = In order to gain an impression of the degree of a sample's reactivity, the ratio between sample and borderline OD is calculated: ratio = OD sample / OD borderline Example: OD borderline = OD sample = ratio = / = INTERPRETATION OF RESULTS Interpretation Qualitative evaluation Ratio negative < 0.80 cut-off 1.0 equivocal Positive > 1.25 The results themselves should not be the only reason for any therapeutical consequences. They have to be correlated to other clinical observations and diagnostic tests. 15. EXPECTED VALUES In a sera collective of 80 blood donors, equally distributed by sex and age, the following distribution of the IgG antibodies was determined: para- number mean mean mean median 95th diagnostic meter of sera + s + 2s percentile specificity dsdna 80 0,28 0,51 0,75 0,21 0,63 98 % RNP 80 0,32 0,45 0,57 0,29 0, % Sm 80 0,18 0,21 0,23 0,18 0, % SS-A/Ro 80 0,25 0,30 0,35 0,25 0, % SS-B/La 80 0,31 0,59 0,87 0,24 0,67 98 % Scl ,40 0,61 0,83 0,33 0,72 98 % CENP-B 80 0,25 0,33 0,41 0,23 0, % Jo ,28 0,40 0,51 0,25 0,43 99 % 16. LIMITATIONS OF THE PROCEDURE Specimen collection has a significant effect on the test results. See SPECIMEN COLLECTION AND STORAGE for details. Azide and thimerosal at concentrations > 0.1 % interfere in this assay and may lead to false results. V / 7

7 17. PERFORMANCE Analytical Sensitivity Detection limit (3 x SD of sample buffer) < 0.2 (ratio; n = 12) Specific for human IgG against dsdna, RNP, Sm, Ro/SS-A, La/SS-B, Scl-70, CENP-B Analytical Specificity and Jo-1 Ratio and coefficient are given as mean of all 8 antigens: Intra-assay variability (n = 3) Inter-assay variability (n = 9) Ratio % CV Ratio % CV Precision Operator to operator variability (n = 2) Variability between 2 kit lots (n = 2) Ratio % CV Ratio % CV AUTOMATION Manually versus Dynex DS2 Variability: Using specimen of one and the same kit lot, the variability of assay results were compared between manual operation and the Dynex DS2 automated ELISA system: Intra-Assay Variability (n = 3) CV Mean manual operation Sample Dynex DS2 Sample 1.4 % 1.3 % Inter-Assay Variability (n = 9) CV Mean 1.6 % 2.0 % V / 7

8 19. PRODUCT LITERATURE REFERENCES 1. Nakamura, R. M., Tan, E. M.: Update on autoantibodies to intracellular antigens in systemic rheumatic diseases. Clin Lab Med 12 (1992), Guma, M., Keil, L. B.: Autoantibodies to cellular antigens in systemic autoimmune diseases. J Clin Immunoassay 17 (1994), Fritzler, M. J.: Clinical relevance of autoantibodies in systemic rheumatic diseases. Mol Biol Rep 23 (1996), Hietarinta, M., Lassila, O.: Clinical significance of antinuclear antibodies in systemic rheumatic disease. Ann Med 28 (1996), Messinger, M.: Autoantikörper bei systemischen entzündlich-rheumatischen Erkrankungen (Kollagenosen). In: L. Thomas (ed.): Labor und Diagnose (2005), TH-Books-Verlags-Gesellschaft, Frankfurt/Main, Sommer, R., and Eitelberger, F.: Wertigkeit der Gliadin-Antikörper im Serum zur Diagnose der Zöliakie. Wien Klin Wochenschr 104/4 (1992), V / 7

9 Symbols / Symbole / Symbôles / Símbolos / Símbolos / Σύμβολα REF LOT CONC LYO IVD Cat.-No.: / Kat.-Nr.: / No.- Cat.: / Cat.-No.: / N.º Cat.: / N. Cat.: / Αριθμός-Κατ.: Lot-No.: / Chargen-Bez.: / No. Lot: / Lot-No.: / Lote N.º: / Lotto n.: / Αριθμός -Παραγωγή: Use by: / Verwendbar bis: / Utiliser à: / Usado por: / Usar até: / Da utilizzare entro: / Χρησιμοποιείται από: No. of Tests: / Kitgröße: / Nb. de Tests: / No. de Determ.: / N.º de Testes: / Quantità dei tests: / Αριθμός εξετάσεων: Concentrate / Konzentrat / Concentré / Concentrar / Concentrado / Concentrato / Συμπύκνωμα Lyophilized / Lyophilisat / Lyophilisé / Liofilizado / Liofilizado / Liofilizzato / Λυοφιλιασμένο In Vitro Diagnostic Medical Device. / In-vitro-Diagnostikum. / Appareil Médical pour Diagnostics In Vitro. / Dispositivo Médico para Diagnóstico In Vitro. / Equipamento Médico de Diagnóstico In Vitro. / Dispositivo Medico Diagnostico In vitro. / Ιατρική συσκευή για In-Vitro ιάγνωση. Evaluation kit. / Nur für Leistungsbewertungszwecke. / Kit pour évaluation. / Juego de Reactivos para Evaluació. / Kit de avaliação. / Kit di evaluazione. / Κιτ Αξιολόγησης. Read instructions before use. / Arbeitsanleitung lesen. / Lire la fiche technique avant emploi. / Lea las instrucciones antes de usar. / Ler as instruções antes de usar. / Leggere le istruzioni prima dell uso. / ιαβάστε τις οδηγίες πριν την χρήση. Keep away from heat or direct sun light. / Vor Hitze und direkter Sonneneinstrahlung schützen. / Garder à l abri de la chaleur et de toute exposition lumineuse. / Manténgase alejado del calor o la luz solar directa. / Manter longe do calor ou luz solar directa. / Non esporre ai raggi solari. / Να φυλάσσεται μακριά από θερμότητα και άμεση επαφή με το φως του ηλίου. Store at: / Lagern bei: / Stocker à: / Almacene a: / Armazenar a: / Conservare a: / Αποθήκευση στους: Manufacturer: / Hersteller: / Fabricant: / Productor: / Fabricante: / Fabbricante: / Παραγωγός: Caution! / Vorsicht! / Attention! / Precaución! / Cuidado! / Attenzione! / Προσοχή! Symbols of the kit components see MATERIALS SUPPLIED. Die Symbole der Komponenten sind im Kapitel KOMPONENTEN DES KITS beschrieben. Voir MATERIEL FOURNI pour les symbôles des composants du kit. Símbolos de los componentes del juego de reactivos, vea MATERIALES SUMINISTRADOS. Para símbolos dos componentes do kit ver MATERIAIS FORNECIDOS. Per i simboli dei componenti del kit si veda COMPONENTI DEL KIT. Για τα σύμβολα των συστατικών του κιτ συμβουλευτείτε το ΠΑΡΕΧΟΜΕΝΑ ΥΛΙΚΑ. COMPLAINTS: Complaints may be submitted initially written or vocal. Subsequently they need to be filed including the test performance and results in writing in case of analytical reasons. WARRANTY: The product is warranted to be free from material defects within the specific shelf life and to comply with product specifications delivered with the product. The product must be used according to the Intended use, all instructions given in the instructions for use and within the product specific shelf life. Any modification of the test procedure or exchange or mixing of components of different lots could negatively affect the results. These cases invalidate any claim for replacement. LIMITATION OF LIABILITY: IN ALL CIRCUMSTANCES THE EXTENT OF MANUFACTURER S LIABILITY IS LIMITED TO THE PURCHASE PRICE OF THE KIT(S) IN QUESTION. IN NO EVENT SHALL MANUFACTURER BE LIABLE FOR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING DAMAGES FOR LOST PROFITS, LOST SALES, INJURY TO PERSON OR PROPERTY OR ANY OTHER INCIDENTAL OR CONSEQUENTIAL LOSS. IBL International GmbH Flughafenstr. 52A, Hamburg, Germany Tel.: + 49 (0) Fax: IBL@IBL-International.com WEB: Symbols Version 4.5 /